VEGF Receptor Blockade Markedly Reduces Retinal Microglia/Macrophage Infiltration into Laser-Induced CNV
Vinores SA (2013) VEGF Receptor Blockade Markedly Reduces Retinal Microglia/Macrophage Infiltration into
Laser-Induced CNV. PLoS ONE 8(8): e71808. doi:10.1371/journal.pone.0071808
VEGF Receptor Blockade Markedly Reduces Retinal Microglia/Macrophage Infiltration into Laser-Induced CNV
Hu Huang 0
Rachel Parlier 0
Ji-kui Shen 0
Gerard A. Lutty 0
Stanley A. Vinores 0
Michael E. Boulton, Indiana University College of Medicine, United States of America
0 Wilmer Eye Institute, Johns Hopkins University School of Medicine , Baltimore, Maryland , United States of America
Although blocking VEGF has a positive effect in wet age-related macular degeneration (AMD), the effect of blocking its receptors remains unclear. This was an investigation of the effect of VEGF receptor (VEGFR) 1 and/or 2 blockade on retinal microglia/macrophage infiltration in laser-induced choroidal neovascularization (CNV), a model of wet AMD. CNV lesions were isolated by laser capture microdissection at 3, 7, and 14 days after laser and analyzed by RT-PCR and immunofluorescence staining for mRNA and protein expression, respectively. Neutralizing antibodies for VEGFR1 or R2 and the microglia inhibitor minocycline were injected intraperitoneally (IP). Anti-CD11b, CD45 and Iba1 antibodies were used to confirm the cell identity of retinal microglia/macrophage, in the RPE/choroidal flat mounts or retinal cross sections. CD11b(+), CD45(+) or Iba1(+) cells were counted. mRNA of VEGFR1 and its three ligands, PlGF, VEGF-A (VEGF) and VEGF-B, were expressed at all stages, but VEGFR2 were detected only in the late stage. PlGF and VEGF proteins were expressed at 3 and 7 days after laser. Anti-VEGFR1 (MF1) delivered IP 3 days after laser inhibited infiltration of leukocyte populations, largely retinal microglia/macrophage to CNV, while anti-VEGFR2 (DC101) had no effect. At 14 days after laser, both MF1 and DC101 antibodies markedly inhibited retinal microglia/macrophage infiltration into CNV. Therefore, VEGFR1 and R2 play differential roles in the pathogenesis of CNV: VEGFR1 plays a dominant role at 3 days after laser; but both receptors play pivotal roles at 14 days after laser. In vivo imaging demonstrated accumulation of GFP-expressing microglia into CNV in both CX3CR1gfp/gfp and CX3CR1gfp/+ mice. Minocycline treatment caused a significant increase in lectin+ cells in the subretinal space anterior to CNV and a decrease in dextran-perfused neovessels compared to controls. Targeting the chemoattractant molecules that regulate trafficking of retinal microglia/macrophage appears to be a compelling therapeutic strategy to control CNV and treat wet AMD.
-
Funding: Supported by National Institutes of Health grants RO1-EY017164 (to SAV and GAL), RO1-EY016557 (to GAL), EY001765 (to Wilmer), Unrestricted funds
from research to Prevent Blindness (to Wilmer), and a stipend from Lilly/ImClone. The authors also thank the Raab Family Foundation and the Wilmer Pooled
Professors Fund for the purchase of the Micron III. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
manuscript.
Competing Interests: Lilly/ImClone provided funding towards this study. There are no patents, products in development or marketed products to declare. This
does not alter the authors adherence to all the PLOS ONE policies on sharing data and materials.
Choroidal neovascularization (CNV) occurs in exudative or wet
age-related macular degeneration (AMD) [1]. The new abnormal
blood vessels in CNV sprout from pre-existing choroidal vessels,
grow through Bruchs membrane, and invade the sub-retinal space
between the pigmented epithelium (RPE) and the photoreceptor
outer segments. Invasion of CNV into the sub-retinal space can
cause pathological consequences, including retinal edema,
detachment and hemorrhage [2]. CNV development in AMD patients
can be characterized into three distinct stages: early/initiation,
intermediate/active, and late/involution [3]. The causative factors
that trigger CNV formation and the cascades of events during the
pathogenesis of CNV are poorly understood, but epidemiological
and experimental evidence suggest several risk factors that are
associated with CNV formation: genetic pre-disposition,
hypertension, cigarette smoking, excessive light exposure, and aging [4
7]. To elucidate the mechanisms regulating the pathogenesis of
CNV, experimental CNV has been generated in various animal
species. The approaches that have been used to create CNV
include subretinal deposit of high molecular weight materials, such
as matrigel [8] and polyethylene glycol [9], oxidized lipid [10], and
laser injury [11,12]. The first two can be considered comparable to
the aberrant deposits of extracellular substance in the sub-retinal
space, similar to that present in AMD patients; the third is initiated
by damage to Bruchs Membrane and RPE. Despite the
differences, both approaches create a microenvironment fostering
CNV or angiogene (...truncated)